1. Field of the Invention
This invention relates to the use of a template-dependent in vitro system for the replication of double stranded genomic RNA on mRNA templates. This is a major advance that will allow the study and definition of replication signals on the template RNA, and packaging signals in viral packaging complexes and on the packaged mRNA. Second, since this system is template dependent, it allows the manipulation of the input mRNAs so that transcripts made from cDNA clones of double stranded RNA virus genes can be included in the system and replicated. Accordingly, this invention also relates to the rescue of exogenous genes into the genomes of double stranded RNA viruses.
2. Description of the Related Technology
The double stranded RNA (dsRNA) viruses are a large and diverse group, encompassing such dissimilar viruses as [i] the L-A virus of yeast which contains a single chromosome, [ii] the two segmented members of the Birnaviridae, [iii] the lipid-containing bacteriophage .phi.6 which has a genome of three segments, and [iv] the members of the Reoviridae (reovirus, rotavirus, orbivirus, etc) which contain a genome of 10-12 segments. More recently, single chromosome dsRNA viruses of parasitic protozoans and the Chestnut blight fungus Cryhonectria parasitica have been characterized.
Among the Reoviridae, rotaviruses are documented as the major cause of diarrheal disease in children and the young of mammalian and avian species. In the United States, acute viral gastroenteritis is a common illness affecting all age groups and is second in frequency only to respiratory illness. In the US, the disease is usually self-limiting although it can be lethal in elderly, debilitated, immunocompromised (including AIDS and transplant), or infant patients. In developing areas of the world, diarrhea disease ranks first in both disease incidence and severity, and it has been estimated that in these regions 3-5 billion cases of diarrhea account for 5-10 million diarrhea-associated deaths annually. Rotaviruses are also recognized as significant veterinary pathogens. Thus, the rotaviruses are important human and animal pathogens that cause significant morbidity and mortality.
In the life cycle of rotavirus, the parental genetic material (11 segments of double-stranded RNA) always remains associated with a subviral particle, the transcriptase particle. The genetic information is transmitted to progeny viruses via 11 (+)-sense or messenger RNAs that are single-stranded and are produced by the transcriptase particle from each of the 11 viral genome segments. The mRNA is translated to make viral proteins. Specific viral proteins join with the 11 mRNAs to form complexes called replicase particles. In the replicase particle, the 11 single stranded mRNAs are replicated to form progeny dsRNA genomes with 11 segments of dsRNA. After the dsRNA is formed, non-structural proteins leave the particle and additional viral structural proteins are added to the replicase particle to complete the morphogenesis of the progeny virus particle.
These viruses enter the host cell and replicate in the cytoplasm. After removal of capsid proteins, which activates the virion transcriptase, single-stranded RNA (ssRNA) of messenger or plus (+)-sense is made and expelled from the particle into the cytoplasm (transcription). These ssRNAs can function as message for the synthesis of protein or as template upon which progeny dsRNA genomes are made by synthesis of the complementary negative sense strand. This latter process is traditionally called replication and the enzyme catalyzing (-)-sense RNA synthesis is called the replicase. Relatively little is known about how replication occurs, but studies of replicase complexes isolated from infected cells show that complexes are formed that contain viral structural and nonstructural proteins and a complete set of the (+)-sense template RNAs. After formation of these particulate structures, the replicase is activated and minus (-)-strand synthesis occurs. Perhaps the most unusual feature of this replication pathway is that both transcription and replication occur in particles from which active enzymes have not been solubilized.
Although a great deal has been learned of rotavirus genome structure (the entire genome of 11 segments has been cloned, sequenced and expressed), predicted protein structure, virion structure, antigenic structure and serology, and genetics (FIG. 1), there are still a number of basic questions related to rotavirus biology and molecular biology which remain unanswered, in particular, what are the molecular bases of genome segment assortment and genome replication. With respect to genome segment assortment, researchers are searching for answers to the following questions: What is the mechanism by which genome segments are selected and packaged so that the progeny virions each contain a complete set of genome segments? What are the cis-acting signals on the (+)-strand RNA that direct their binding/packaging by the particulate replicase? What component of the particle binds the binding site on the (+)-strand RNA? What are the packaging requirements for completion of morphogenesis of the subviral particle (SVP) that has segregated a complete set of genome segments? Is the packaging reaction selective for viral (+)-strand RNA, or will it package any ssRNA?
With respect to genome replication, researchers are searching for answers to the following questions: What are the cis-acting signals on the (+)-strand RNA that direct synthesis of the (-)-strand by the replicase? Are there packaging requirements that must be fulfilled for the replicase to be activated? What are the optimum conditions and requirements of the replicase enzyme? If nonviral ssRNAs are packaged, are they replicated? If not, what are the signals for replication of virus-specific (+)-strand RNAs?
The answers to these questions will enhance the understanding of the molecular mechanisms by which complex viruses replicate and interact with the host cell, and will reveal the mechanisms characteristic of rotavirus that will be useful in development of disease control strategies, vaccines and antivirals.
A major stumbling block to finding the answers to these questions and to defining the roles of the various components of the replicase particles has been the absence of a template-dependent in vitro RNA replication system for the Reoviridae. Prior to the present invention, no template-dependent replication system has been described and exploited for the study of RNA replication in any member of the Reoviridae.
Another major stumbling-block to progress in molecular analyses of the Reoviridae is the absence of a system that allows the "rescue" of (+)-strand RNA transcripts of cloned and manipulated cDNAs into infectious virus. A system that could potentially lead to rescue in rotavirus was recently published, reporting replication of exogenously added (+)-strand RNA by replicase particles isolated from rotavirus-infected cells was made (Gorziglia, M. and Collins, P. (1992), Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene. Proc. Natl. Acad. Sci. 89:5784-5788), but this report has not been followed-up. This system is typical of the approach used by many rotavirus labs, without success.
According to this approach, virus-infected tissue culture cells perform the "work" of rescue of an exogenous rotavirus gene (or synthetic gene that looks to the cell like a rotavirus gene) into progeny virus particles. However, the cells used in this approach are not normal cells. These cells have been genetically engineered so that they are expressing a precise copy of a single rotavirus mRNA, even though those cells are not infected. After it is established that the cells are expressing the exogenous rotavirus mRNA, the cells are infected with an ordinary rotavirus using standard conditions for infection. When the infecting rotavirus enters the cell, it produces the 11 rotavirus mRNAs from the transcriptase particle. These 11 "native" rotavirus mRNAs can mix in the cell with the mRNA from the exogenous rotavirus gene. At the stage of the life cycle where viral proteins and mRNAs form replicase particles, it is hoped that by chance the particle will contain 10 of the "native" mRNAs and that the 11th mRNA will the rotavirus mRNA expressed by the cell rather than the equivalent mRNA made by the infecting virus. If this happens, after replication and completion of the life cycle, the progeny virus particle will contain 10 genome segments derived from the infecting virus and the 11th genome segment derived from the exogenous rotavirus gene expressed by the cells. This would constitute rescue of the exogenous gene into infectious virus. The whole process is carried out in the context of the infected cell, and utilizes the normal viral infectious pathways for the life cycle. It is by chance that the extra rotavirus mRNA present in the infected cell becomes incorporated (or rescued) into the progeny virus. There are no reported successes using this system.
The present invention overcomes both of the above-mentioned stumbling blocks and represents a powerful tool for the research of rotavirus and the Reoviridae family of viruses by providing both a system for in vitro replication and a method for the rescue of exogenous genes into infections virus particles.